NON-IONIC DETERGENTS
NON-IONIC DETERGENTS
Non-ionic Detergents
NON-IONIC DETERGENTS OFFERED BY BACHEM Detergents are amphipathic molecules that contain both polar and hydrophobic groups. All detergents are characterized as containing a hydrophilic “head” region and a hydrophobic “tail” region. In contrast to purely polar or non-polar molecules, amphipathic molecules exhibit unique properties in water. Their polar group forms hydrogen bonds with water molecules, while the hydrocarbon chains aggregate due to hydrophobic interactions. These properties allow detergents to be soluble in water and also to solubilize hydrophobic compounds in water.
NON-IONIC DETERGENTS Bachem offers alkanoyl-N-methylglucamides (MEGA’s), alkylglycosides, and oligoethyleneglycol monoalkyl ethers. Besides these types of nonionic detergents, a choice of lipids and phospholipids can be ordered at our online shop: shop.bachem.com
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Overview Detergents Detergents are used in biochemistry to solubilize membrane-bound proteins and facilitate their isolation and recrystallization [1,2]. They can be used to introduce the purified protein molecules into liposomes and to determine their properties or enzymatic activities in reconstitution experiments [3,4]. Detergents can further be used to facilitate protein solubilization in biological assays or to form microemulsions for electrochemical studies [5]. There are hundreds of detergents that can be used in biochemical experiments. The selection of the “right” detergent for a given application is therefore difficult and still rather empirical [1,3,6,7]. The following physical properties of detergents can help making the right decision. Critical Micelle Concentration (CMC) The CMC of a detergent is the minimum concentration at which micelles form. The CMC is also an indicator of the strength detergents bind to proteins; i.e., low values indicate strong binding and high values indicate weak binding. The CMC is influenced by pH, temperature, ionic strength and impurities in the solution [1,7,8]. The CMC values reported in literature (e.g. [9]) are therefore only correct for the given conditions. • A high CMC is desirable when dialysis across a membrane is necessary and in other situations where rapid removal or displacement of detergent is desired. • A low CMC is desirable if the ratio of free to bound detergent has to be minimized, e.g. in the measurement of binding strength of detergent to protein. • The CMC of a given detergent can be determined by different methods including the measurement of light scattering, surface tension, hydrodynamic properties, and changes in absorbance or fluorescence upon dye solubilization [10].
CnEm
Product
C5E1
Ethyleneglycolmonopentylether
C5E2
Diethyleneglycolmonopentylether
C5E3
n-Pentyltrioxyethylene
C6E1
Ethyleneglycolmonohexylether
C6E3
n-Hexyltrioxyethylene
C6E4
n-Hexyltetraoxyethylene
C6E5
n-Hexylpentaoxyethylene
C7E3
n-Heptyltrioxyethylene
C7E4
n-Heptyltetraoxyethylene
C7E5
n-Heptylpentaoxyethylene
C8E1
Ethyleneglycolmonooctylether
C8E3
n-Octyltrioxyethylene
C8E4
n-Octyltetraoxyethylene
C8E5
n-Octylpentaoxyethylene
C8En
n-Octylpolyoxyethylene
C10E4
n-Decyltetraoxyethylene
C10E5
n-Decylpentaoxyethylene
C12E5
Dodecyl pentaethylene glycolether
n-Alkyloligooxyethylenes (Oligoethyleneglycol monoalkyl ethers) CnEm: Cn = CH3(CH2)n-1 alkyl chain Em = (OCH2CH2)mOH oligoethyleneglycol In particular, the octyl oligooxyethylenes C8E4, C8E5, and C8En (OctylPOE, n = 2 to 9) have been shown to be of great value for solubilization and crystallization of proteins, such as porin, from E. coli outer membranes.
Aggregation Number (N) The aggregation number is the average number of detergent monomers in one micelle and permits the determination of the micelle size and the molecular weight. The micelle size is important in gel filtration experiments since the separation of different proteins according to size is done more easily in the presence of a detergent with a small micellar size [6]. This value is dependent on temperature and ionic strength. Cloud Point (cp) The cloud point is the temperature above which detergent micelles form superaggregates and the solution separates in a solvent-rich phase and a solvent-depleted phase [7]. This phase separation can be exploited for protein extraction.
3
Non-ionic Detergents
MILD NON-IONIC DETERGENTS FOR THE SOLUBILIZATION OF MEMBRANE-BOUND PROTEINS Hydrophilic head
Hydrophobic tail
Critical Micellar Temperature (cmt) The critical micellar temperature is defined as the minimum temperature at which a detergent can form micelles in water. At temperatures below cmt, some detergents exist as insoluble liquid crystals. This parameter has to be taken into consideration in protein purification at low temperatures. Classification Detergents can be grouped in four main classes according to the properties of their head group: • Anionic • Cationic • Zwitterionic (Ampholytic) • Non-Ionic Anionic and cationic detergents typically modify protein structure to a greater extent than the other two classes. Zwitterionic detergents are unique as they offer the combined properties of ionic and non-ionic detergents. Like non-ionic detergents the zwittergents do not possess a net charge, they lack conductivity and electrophoretic mobility and do not bind
4
to ion-exchange resins. However, like ionic detergents, they are efficient at breaking protein-protein interactions. The group of non-ionic detergents, presented in this brochure, is special in the sense that they can be classified as “mild” detergents because they are less likely to denature proteins than ionic detergents. On the other side they are less effective at disrupting protein aggregation. The most important properties of non-ionic detergents are: • Uncharged hydrophilic head group. • Better suited for breaking lipid-lipid and lipid-protein interactions. • Considered to be nondenaturants. • Salts have minimal effect on micellar size. • Solubilize membrane proteins in a gentle manner, allowing the solubilized proteins to retain native subunit structure, enzymatic activity and/or nonenzymatic function. • The CMC of a non-ionic detergent is relatively unaffected by increasing ionic strength, and increases substantially with rising temperature.
The amphipathic nature of detergents is shown as an example with n-Dodecyl-βD-maltoside (DDM, Product P-1170). Maltose constitutes the hydrophilic head and the alkyl chain the hydrophobic tail.
Practical Considerations Denaturation Effect When characterizing a protein in its native state and studying its functions, the denaturation effect of detergents is an important point. It is difficult to classify detergents into denaturizing and non-denaturizing classes and to ascribe these properties to specific features of either the monomeric or the micellar structure of the molecule [8]. The denaturation effect of a detergent depends further on the structure of the protein itself. The following general statements can be of practical use: • Non-ionic detergents with polyoxyethylene or sugar head groups do usually not denaturize proteins. • Ionic detergents are nearly always denaturants at temperatures and concentrations used for complete membrane solubilization. They further dissociate complex proteins in their polypeptide chains. This effect may be useful in the separation and identification of the different subunits of a protein [6]. Detergent Amount / Concentration The appropriate amount of detergent (concentration) must be used for a successful isolation of a protein. The membranes undergo different stages of disintegration with an increasing amount of detergent: • At concentrations of around 0.1 mg to 1 mg detergent per mg membrane lipid, selective extraction of membrane proteins can occur but the membrane bilayer remains essentially intact. • At higher concentrations of about 2 mg detergent per mg lipid, solubilization of the membrane occurs. This results in the formation of soluble lipid-protein-detergent, protein-detergent and lipid-detergent micelles [1]. • 10 mg detergent per mg lipid or more should be used for delipidation (i.e. a maximal exchange of the lipid bound to the protein with detergent). At this point protein-detergent micelles are formed, each containing essentially only one protein molecule. These micelles can then be separated by methods based on size, charge density, binding affinity and solvent partitioning [11]. In the solubilization process the binding of detergent to protein or membranes (solubi-
lization) has to compete with the self-association of detergent molecules to micelles [1,12]. The exact amount of detergent needed to achieve a certain effect depends on the CMC, the micelle size, the temperature, the nature of the membrane and the detergent [1]. The detergent monomers do not participate in membrane solubilization, but they are required to obtain the monomer-micelle equilibrium [11]. To calculate the effective detergent-to-lipid ratio and the amount of detergent available for solubilization, the amount of detergent forming micelles must be taken into account: • Detergents with low CMC; the effective amount of detergent essentially equals the total amount of detergent added, since very little detergent exists as monomers. • Detergents with a high CMC; the effective amount of detergent equals the total amount of detergent added minus the monomer concentration (essentially the CMC).
Despite the large number of detergents that are commercially available, no single “universal detergent” is ideally suited to all biochemical applications. (G.G.Privé)
References [1] A. Helenius and K. Simons, Biochim. Biophys. Acta 415, 29 (1975) [2] R.M. Garavito and J.A. Jenkins, Structure and Function of Membrane Proteins (E. Quagliariello and F. Palmieri, eds.), Elsevier Science Publishers B.V., Amsterdam (1983) [3] C. Tanford and J.A. Reynolds, Biochim. Biophys. Acta 457, 133 (1976) [4] M. Hanatani et al., J. Biochem. 95, 1349 (1984) [5] M.O. Iwunze et al., Anal. Chem. 62, 644 (1990) [6] A. Helenius et al., Methods Enzymol. 56, 734 (1979) [7] J.M. Neugebauer, Methods Enzymol. 182, 239 (1990) [8] L.M. Hjelmeland, Methods Enzymol. 124, 135 (1986) [9] P. Mukerjee and K.J. Mysels, National Standards Reference Data Series, Vol. 36, US National Bureau of Standards (NSRDSNBS 36), Washington (1971) [10] A. Chattopadhyay and E. London, Anal. Biochem. 139, 408 (1984) [11] D. Lichtenberg et al., Biochim. Biophys. Acta 737, 285 (1983) [12] C. Tanford, J. Mol. Biol. 67, 59 (1972)
5
Non-ionic Detergents
GENERAL REFERENCES C. Tanford The hydrophobic effect: Formation of micelles and biological membranes. Second edition. John Wiley and Sons, New York (1980) U. Pfüller Mizellen - Vesikel- Microemulsionen. Tensidassoziate und ihre Anwendung in Analytik und Biochemie. Springer-Verlag, Berlin (1986) H. Michel Crystallization of membrane proteins. CRC Press Inc., Boca Raton (1991) E.D. Goddard and K.P. Ananthapadman-anbhan, eds. Interactions of surfactants with polymers and proteins. CRC Press Inc., Boca Raton (1993) K. Holmberg, ed. Novel surfactants: Preparation, applications and biodegradability. Second edition. Marcel Dekker Inc., New York (2003)
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M. Caffrey Membrane protein crystallization. J. Struct. Biol. 142, 108-132 (2003) M.C. Wiener A pedestrian guide to membrane protein crystallization. Methods 34, 364-372 (2004) D. Myers Surfactants science and technology. Third edition. John Wiley and Sons, New York (2006) G.G. Privé Detergents for the stabilization and crystallization of membrane proteins. Methods 41, 388-397 (2007)
NON-IONIC DETERGENTS Bilayers and Micelles. Detergents are amphipathic molecules that contain both polar and hydrophobic groups. All detergents are characterized as containing a hydrophilic “head” region and a hydrophobic “tail” region. In contrast to purely polar or non-polar molecules, amphipathic molecules exhibit unique properties in water. Their polar group forms hydrogen bonds with water molecules, while the hydrocarbon chains aggregate due to hydrophobic interactions. These properties allow detergents to be soluble in water and also to solubilize hydrophobic compounds in aqueous systems.
7
Non-ionic Detergents
NON-IONIC DETERGENTS Alkanoyl-N-methylglucamides combine a high solubilization power with non-denaturizing properties. They don’t interfere with the photometric monitoring of proteins at 280 nm (as their absorption maximum lies at 220 nm). Their easy removal by dialysis makes them valuable tools for membrane studies. Like the alkanoyl-N-methylglucamides, the alkylglucosides are mild detergents. Their CMC are only slightly affected by variations of ionic strength. Oligoethyleneglycol monoalky ethers are standard detergents for the solubilization and structural characterization of integral membrane proteins.
N-D-GLUCON-METHYLALKANAMIDES (MEGAs)
ALKYL GLYCOSIDES 8
MEGA-8 (Octanoyl-N-methylglucamide) P-1060
MEGA-10 (Decanoyl-N-methylglucamide) P-1000
MEGA-9 (Nonanoyl-N-methylglucamide) P-1165
MEGA-12 (Dodecanoyl-N-methylglucamide) P-1175
n-Nonyl β-D-glucopyranoside (NGP) P-1150
n-Dodecyl-β-D-maltoside (DDM) P-1170
Octyl glucoside (OGP) P-1110
OLIGOETHYLENEGLYCOL MONOALKYL ETHERS AND SULFOXIDES
Ethyleneglycolmonopentylether (C5E1; n-Pentylmonooxyethylene) P-1055
Ethyleneglycolmonooctylether (C8E1; n-Octylmonooxyethylene) P-1050
Diethyleneglycolmonopentylether (C5E2; n-Pentyldioxyethylene) P-1025
n-Octyltrioxyethylene (C8E3; Triethyleneglycolmonooctyl ether) P-1125
n-Pentyltrioxyethylene (C5E3; Triethyleneglycolmonopentyl ether) P-1135
n-Octyltetraoxyethylene (C8E4; Tetraethyleneglycolmonooctyl ether) P-1120
Ethyleneglycolmonohexylether (C6E1; n-Hexylmonooxyethylene) P-1045
n-Octylpentaoxyethylene (C8E5; Pentaethyleneglycolmonooctyl ether) P-1115
n-Hexyltrioxyethylene (C6E3; Triethyleneglycolmonohexyl ether) P-1095 n-Hexyltetraoxyethylene (C6E4; Tetraethyleneglycolmonohexyl ether) P-1085 n-Hexylpentaoxyethylene (C6E5; Pentaethyleneglycolmonohexyl ether) P-1080 n-Heptyltrioxyethylene (C7E3; Triethyleneglycolmonoheptyl ether) P-1075 n-Heptyltetraoxyethylene (C7E4; Tetraethyleneglycolmonoheptyl ether) P-1070 n-Heptylpentaoxyethylene (C7E5; Pentaethyleneglycolmonoheptyl ether) P-1065
n-Octylpolyoxyethylene (C8En (n = 2 to 9); Octyl-POE; Rosenbusch-Tenside) P-1140 n-Decyltetraoxyethylene (C10E4; Tetraethyleneglycolmonodecyl ether) P-1010 n-Decylpentaoxyethylene (C10E5; Pentaethyleneglycolmonodecyl ether) P-1005 Dodecyl pentaethyleneglycolether (C12E5; Pentaethyleneglycolmonododecyl ether) P-1160 rac-2,3-Dihydroxypropyloctylsulfoxide (n-Octyl-rac-2,3-dioxypropyl sulfoxide) P-1040 2-Hydroxyethyloctylsulfoxide (n-Octyl-2-hydroxyethyl sulfoxide) P-1105
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Non-ionic Detergents
Product Number
Product
CMC (mM)
Conditions
N (H2O)
References
P-1060
MEGA-8
73
25°C
F. Yu and R.E. McCarty, Arch. Biochem. Biophys. 238, 61 (1985)
P-1165
MEGA-9
25
25°C
V. de Pinto et al. , Eur. J. Biochem. 183, 179 (1989)
50
15°C
P-1000
MEGA-10
7
P-1175
MEGA-12
0.35
25°C
Y.-P. Zhu et al., J. Surf. Det. 2, 357 (1999)
P-1150
n-Nonyl β-Dglucopyranoside
6.5
low ionic strength 133
W.J. de Grip and P.H.M. Bovee-Geurts, Chem. Phys Lipids 23, 321 (1979)
3.5
1M NaCl
7.5
25°C
F. Yu and R.E. McCarty, Arch. Biochem. Biophys. 238, 61 (1985)
23.2
W.J. de Grip and P.H.M. Bovee-Geurts, Chem. Phys Lipids 23, 321 (1979)
13.5
low ionic strength 84 27-100 1M NaCl
25.0
25°C
K. Shinoda et al., Bull. Chem. Soc. Jpn. 34, 237 (1961)
P-1110
Octyl glucoside
M. Hanatani et al., J. Biochem. 95, 1349 (1984)
P-1170
n-Dodecyl-β-Dmaltoside
170
78-149 98
P-1095
n-Hexyltrioxyethylene
100
25°C
P-1085
n-Hexyltetraoxyethylene
90
20°C
P-1080
n-Hexylpentaoxyethylene
90
20°C
P-1050
Ethyleneglycolmonooctyl ether
4.9
25°C
P-1125
n-Octyltrioxyethylene
7.5
25°C
P-1120
n-Octyltetraoxyethylene
12.4
6°C
3.6
60°C
M. Corti et al., Phys. Rev. Lett. 48, 1617 (1982) R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986)
25°C
M. Zulauf and J.P. Rosenbusch, J. Phys. Chem. 87, 856 (1983)
P-1115
n-Octylpentaoxyethylene
4.3
P-1140
n-Octylpolyoxyethylene
6.6
P-1010
n-Decyltetraoxyethylene
0.98
10°C
0.68
25°C
P-1005
n-Decylpentaoxyethylene
1.18
10°C
0.81
25°C
P-1105
10
2-Hydroxyethyloctyl sulfoxide
29.9
W.J. de Grip, Methods Enzymol. 81, 256 (1982) J. Kern et al., Photosynth. Res. 84, 153 (2005) P. Becher, Micelle formation in aqueous and nonaqueous solutions. In: Nonionic Surfactants, M.J. Schick, ed., Marcel Dekker Inc., New York, p. 478 (1967)
82
R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986) 53 ± 10
P. Becher, Micelle formation in aqueous and nonaqueous solutions. In: Nonionic Surfactants, M.J. Schick, ed., Marcel Dekker Inc., New York, p. 478 (1967)
73
R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986)
PRODUCT BROCHURES AMYLOID PEPTIDES
ANTIMICROBIAL PEPTIDES
CALCITONIN GENE-RELATED PEPTIDES
CASPASE SUBSTRATES INHIBITORS
CYSTEINE DERIVATIVES
DAP AND DAB DERIVATIVES
DIABETES PEPTIDES
ENDOTHELINS
FRET SUBSTRATES
GHRELIN, LEPTIN AND OBESTATIN
1
1
LHRH AGONISTS AND ANTAGONISTS
MATRIX METALLOPROTEINASES
MELANOMA PEPTIDES
N-METHYLATED AMINO ACID DERIVATIVES
NEUROPEPTIDE Y
NON-IONIC DETERGENTS
ORTHOGONALITY OF PROTECTING GROUPS
PAR ACTIVATING PEPTIDES
PEPTIDE YY
PEPTIDES IN COSMETICS
SECRETASE SUBSTRATES INHIBITORS
VETERINARY PEPTIDES
VIP/PACAP
T
T S
PRION PEPTIDES
PSEUDOPROLINE DIPEPTIDES
Europe, Africa, Middle East and Asia Pacific: Bachem AG Tel. +41 61 935 2323 [email protected] Americas Bachem Americas, Inc. Tel. +1 888 422 2436 [email protected] Visit our website www.bachem. com or shop online shop.bachem.com
All information is compiled to the best of our knowledge. We cannot be made liable for any possible errors or misprints. Some products may be restricted in certain countries.
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shop.bachem.com
Published by Global Marketing, Bachem Group, March 2014
Marketing & Sales Contact
Non-ionic Detergents
NON-IONIC DETERGENTS OFFERED BY BACHEM Detergents are amphipathic molecules that contain both polar and hydrophobic groups. All detergents are characterized as containing a hydrophilic “head” region and a hydrophobic “tail” region. In contrast to purely polar or non-polar molecules, amphipathic molecules exhibit unique properties in water. Their polar group forms hydrogen bonds with water molecules, while the hydrocarbon chains aggregate due to hydrophobic interactions. These properties allow detergents to be soluble in water and also to solubilize hydrophobic compounds in water.
NON-IONIC DETERGENTS Bachem offers alkanoyl-N-methylglucamides (MEGA’s), alkylglycosides, and oligoethyleneglycol monoalkyl ethers. Besides these types of nonionic detergents, a choice of lipids and phospholipids can be ordered at our online shop: shop.bachem.com
2
Overview Detergents Detergents are used in biochemistry to solubilize membrane-bound proteins and facilitate their isolation and recrystallization [1,2]. They can be used to introduce the purified protein molecules into liposomes and to determine their properties or enzymatic activities in reconstitution experiments [3,4]. Detergents can further be used to facilitate protein solubilization in biological assays or to form microemulsions for electrochemical studies [5]. There are hundreds of detergents that can be used in biochemical experiments. The selection of the “right” detergent for a given application is therefore difficult and still rather empirical [1,3,6,7]. The following physical properties of detergents can help making the right decision. Critical Micelle Concentration (CMC) The CMC of a detergent is the minimum concentration at which micelles form. The CMC is also an indicator of the strength detergents bind to proteins; i.e., low values indicate strong binding and high values indicate weak binding. The CMC is influenced by pH, temperature, ionic strength and impurities in the solution [1,7,8]. The CMC values reported in literature (e.g. [9]) are therefore only correct for the given conditions. • A high CMC is desirable when dialysis across a membrane is necessary and in other situations where rapid removal or displacement of detergent is desired. • A low CMC is desirable if the ratio of free to bound detergent has to be minimized, e.g. in the measurement of binding strength of detergent to protein. • The CMC of a given detergent can be determined by different methods including the measurement of light scattering, surface tension, hydrodynamic properties, and changes in absorbance or fluorescence upon dye solubilization [10].
CnEm
Product
C5E1
Ethyleneglycolmonopentylether
C5E2
Diethyleneglycolmonopentylether
C5E3
n-Pentyltrioxyethylene
C6E1
Ethyleneglycolmonohexylether
C6E3
n-Hexyltrioxyethylene
C6E4
n-Hexyltetraoxyethylene
C6E5
n-Hexylpentaoxyethylene
C7E3
n-Heptyltrioxyethylene
C7E4
n-Heptyltetraoxyethylene
C7E5
n-Heptylpentaoxyethylene
C8E1
Ethyleneglycolmonooctylether
C8E3
n-Octyltrioxyethylene
C8E4
n-Octyltetraoxyethylene
C8E5
n-Octylpentaoxyethylene
C8En
n-Octylpolyoxyethylene
C10E4
n-Decyltetraoxyethylene
C10E5
n-Decylpentaoxyethylene
C12E5
Dodecyl pentaethylene glycolether
n-Alkyloligooxyethylenes (Oligoethyleneglycol monoalkyl ethers) CnEm: Cn = CH3(CH2)n-1 alkyl chain Em = (OCH2CH2)mOH oligoethyleneglycol In particular, the octyl oligooxyethylenes C8E4, C8E5, and C8En (OctylPOE, n = 2 to 9) have been shown to be of great value for solubilization and crystallization of proteins, such as porin, from E. coli outer membranes.
Aggregation Number (N) The aggregation number is the average number of detergent monomers in one micelle and permits the determination of the micelle size and the molecular weight. The micelle size is important in gel filtration experiments since the separation of different proteins according to size is done more easily in the presence of a detergent with a small micellar size [6]. This value is dependent on temperature and ionic strength. Cloud Point (cp) The cloud point is the temperature above which detergent micelles form superaggregates and the solution separates in a solvent-rich phase and a solvent-depleted phase [7]. This phase separation can be exploited for protein extraction.
3
Non-ionic Detergents
MILD NON-IONIC DETERGENTS FOR THE SOLUBILIZATION OF MEMBRANE-BOUND PROTEINS Hydrophilic head
Hydrophobic tail
Critical Micellar Temperature (cmt) The critical micellar temperature is defined as the minimum temperature at which a detergent can form micelles in water. At temperatures below cmt, some detergents exist as insoluble liquid crystals. This parameter has to be taken into consideration in protein purification at low temperatures. Classification Detergents can be grouped in four main classes according to the properties of their head group: • Anionic • Cationic • Zwitterionic (Ampholytic) • Non-Ionic Anionic and cationic detergents typically modify protein structure to a greater extent than the other two classes. Zwitterionic detergents are unique as they offer the combined properties of ionic and non-ionic detergents. Like non-ionic detergents the zwittergents do not possess a net charge, they lack conductivity and electrophoretic mobility and do not bind
4
to ion-exchange resins. However, like ionic detergents, they are efficient at breaking protein-protein interactions. The group of non-ionic detergents, presented in this brochure, is special in the sense that they can be classified as “mild” detergents because they are less likely to denature proteins than ionic detergents. On the other side they are less effective at disrupting protein aggregation. The most important properties of non-ionic detergents are: • Uncharged hydrophilic head group. • Better suited for breaking lipid-lipid and lipid-protein interactions. • Considered to be nondenaturants. • Salts have minimal effect on micellar size. • Solubilize membrane proteins in a gentle manner, allowing the solubilized proteins to retain native subunit structure, enzymatic activity and/or nonenzymatic function. • The CMC of a non-ionic detergent is relatively unaffected by increasing ionic strength, and increases substantially with rising temperature.
The amphipathic nature of detergents is shown as an example with n-Dodecyl-βD-maltoside (DDM, Product P-1170). Maltose constitutes the hydrophilic head and the alkyl chain the hydrophobic tail.
Practical Considerations Denaturation Effect When characterizing a protein in its native state and studying its functions, the denaturation effect of detergents is an important point. It is difficult to classify detergents into denaturizing and non-denaturizing classes and to ascribe these properties to specific features of either the monomeric or the micellar structure of the molecule [8]. The denaturation effect of a detergent depends further on the structure of the protein itself. The following general statements can be of practical use: • Non-ionic detergents with polyoxyethylene or sugar head groups do usually not denaturize proteins. • Ionic detergents are nearly always denaturants at temperatures and concentrations used for complete membrane solubilization. They further dissociate complex proteins in their polypeptide chains. This effect may be useful in the separation and identification of the different subunits of a protein [6]. Detergent Amount / Concentration The appropriate amount of detergent (concentration) must be used for a successful isolation of a protein. The membranes undergo different stages of disintegration with an increasing amount of detergent: • At concentrations of around 0.1 mg to 1 mg detergent per mg membrane lipid, selective extraction of membrane proteins can occur but the membrane bilayer remains essentially intact. • At higher concentrations of about 2 mg detergent per mg lipid, solubilization of the membrane occurs. This results in the formation of soluble lipid-protein-detergent, protein-detergent and lipid-detergent micelles [1]. • 10 mg detergent per mg lipid or more should be used for delipidation (i.e. a maximal exchange of the lipid bound to the protein with detergent). At this point protein-detergent micelles are formed, each containing essentially only one protein molecule. These micelles can then be separated by methods based on size, charge density, binding affinity and solvent partitioning [11]. In the solubilization process the binding of detergent to protein or membranes (solubi-
lization) has to compete with the self-association of detergent molecules to micelles [1,12]. The exact amount of detergent needed to achieve a certain effect depends on the CMC, the micelle size, the temperature, the nature of the membrane and the detergent [1]. The detergent monomers do not participate in membrane solubilization, but they are required to obtain the monomer-micelle equilibrium [11]. To calculate the effective detergent-to-lipid ratio and the amount of detergent available for solubilization, the amount of detergent forming micelles must be taken into account: • Detergents with low CMC; the effective amount of detergent essentially equals the total amount of detergent added, since very little detergent exists as monomers. • Detergents with a high CMC; the effective amount of detergent equals the total amount of detergent added minus the monomer concentration (essentially the CMC).
Despite the large number of detergents that are commercially available, no single “universal detergent” is ideally suited to all biochemical applications. (G.G.Privé)
References [1] A. Helenius and K. Simons, Biochim. Biophys. Acta 415, 29 (1975) [2] R.M. Garavito and J.A. Jenkins, Structure and Function of Membrane Proteins (E. Quagliariello and F. Palmieri, eds.), Elsevier Science Publishers B.V., Amsterdam (1983) [3] C. Tanford and J.A. Reynolds, Biochim. Biophys. Acta 457, 133 (1976) [4] M. Hanatani et al., J. Biochem. 95, 1349 (1984) [5] M.O. Iwunze et al., Anal. Chem. 62, 644 (1990) [6] A. Helenius et al., Methods Enzymol. 56, 734 (1979) [7] J.M. Neugebauer, Methods Enzymol. 182, 239 (1990) [8] L.M. Hjelmeland, Methods Enzymol. 124, 135 (1986) [9] P. Mukerjee and K.J. Mysels, National Standards Reference Data Series, Vol. 36, US National Bureau of Standards (NSRDSNBS 36), Washington (1971) [10] A. Chattopadhyay and E. London, Anal. Biochem. 139, 408 (1984) [11] D. Lichtenberg et al., Biochim. Biophys. Acta 737, 285 (1983) [12] C. Tanford, J. Mol. Biol. 67, 59 (1972)
5
Non-ionic Detergents
GENERAL REFERENCES C. Tanford The hydrophobic effect: Formation of micelles and biological membranes. Second edition. John Wiley and Sons, New York (1980) U. Pfüller Mizellen - Vesikel- Microemulsionen. Tensidassoziate und ihre Anwendung in Analytik und Biochemie. Springer-Verlag, Berlin (1986) H. Michel Crystallization of membrane proteins. CRC Press Inc., Boca Raton (1991) E.D. Goddard and K.P. Ananthapadman-anbhan, eds. Interactions of surfactants with polymers and proteins. CRC Press Inc., Boca Raton (1993) K. Holmberg, ed. Novel surfactants: Preparation, applications and biodegradability. Second edition. Marcel Dekker Inc., New York (2003)
6
M. Caffrey Membrane protein crystallization. J. Struct. Biol. 142, 108-132 (2003) M.C. Wiener A pedestrian guide to membrane protein crystallization. Methods 34, 364-372 (2004) D. Myers Surfactants science and technology. Third edition. John Wiley and Sons, New York (2006) G.G. Privé Detergents for the stabilization and crystallization of membrane proteins. Methods 41, 388-397 (2007)
NON-IONIC DETERGENTS Bilayers and Micelles. Detergents are amphipathic molecules that contain both polar and hydrophobic groups. All detergents are characterized as containing a hydrophilic “head” region and a hydrophobic “tail” region. In contrast to purely polar or non-polar molecules, amphipathic molecules exhibit unique properties in water. Their polar group forms hydrogen bonds with water molecules, while the hydrocarbon chains aggregate due to hydrophobic interactions. These properties allow detergents to be soluble in water and also to solubilize hydrophobic compounds in aqueous systems.
7
Non-ionic Detergents
NON-IONIC DETERGENTS Alkanoyl-N-methylglucamides combine a high solubilization power with non-denaturizing properties. They don’t interfere with the photometric monitoring of proteins at 280 nm (as their absorption maximum lies at 220 nm). Their easy removal by dialysis makes them valuable tools for membrane studies. Like the alkanoyl-N-methylglucamides, the alkylglucosides are mild detergents. Their CMC are only slightly affected by variations of ionic strength. Oligoethyleneglycol monoalky ethers are standard detergents for the solubilization and structural characterization of integral membrane proteins.
N-D-GLUCON-METHYLALKANAMIDES (MEGAs)
ALKYL GLYCOSIDES 8
MEGA-8 (Octanoyl-N-methylglucamide) P-1060
MEGA-10 (Decanoyl-N-methylglucamide) P-1000
MEGA-9 (Nonanoyl-N-methylglucamide) P-1165
MEGA-12 (Dodecanoyl-N-methylglucamide) P-1175
n-Nonyl β-D-glucopyranoside (NGP) P-1150
n-Dodecyl-β-D-maltoside (DDM) P-1170
Octyl glucoside (OGP) P-1110
OLIGOETHYLENEGLYCOL MONOALKYL ETHERS AND SULFOXIDES
Ethyleneglycolmonopentylether (C5E1; n-Pentylmonooxyethylene) P-1055
Ethyleneglycolmonooctylether (C8E1; n-Octylmonooxyethylene) P-1050
Diethyleneglycolmonopentylether (C5E2; n-Pentyldioxyethylene) P-1025
n-Octyltrioxyethylene (C8E3; Triethyleneglycolmonooctyl ether) P-1125
n-Pentyltrioxyethylene (C5E3; Triethyleneglycolmonopentyl ether) P-1135
n-Octyltetraoxyethylene (C8E4; Tetraethyleneglycolmonooctyl ether) P-1120
Ethyleneglycolmonohexylether (C6E1; n-Hexylmonooxyethylene) P-1045
n-Octylpentaoxyethylene (C8E5; Pentaethyleneglycolmonooctyl ether) P-1115
n-Hexyltrioxyethylene (C6E3; Triethyleneglycolmonohexyl ether) P-1095 n-Hexyltetraoxyethylene (C6E4; Tetraethyleneglycolmonohexyl ether) P-1085 n-Hexylpentaoxyethylene (C6E5; Pentaethyleneglycolmonohexyl ether) P-1080 n-Heptyltrioxyethylene (C7E3; Triethyleneglycolmonoheptyl ether) P-1075 n-Heptyltetraoxyethylene (C7E4; Tetraethyleneglycolmonoheptyl ether) P-1070 n-Heptylpentaoxyethylene (C7E5; Pentaethyleneglycolmonoheptyl ether) P-1065
n-Octylpolyoxyethylene (C8En (n = 2 to 9); Octyl-POE; Rosenbusch-Tenside) P-1140 n-Decyltetraoxyethylene (C10E4; Tetraethyleneglycolmonodecyl ether) P-1010 n-Decylpentaoxyethylene (C10E5; Pentaethyleneglycolmonodecyl ether) P-1005 Dodecyl pentaethyleneglycolether (C12E5; Pentaethyleneglycolmonododecyl ether) P-1160 rac-2,3-Dihydroxypropyloctylsulfoxide (n-Octyl-rac-2,3-dioxypropyl sulfoxide) P-1040 2-Hydroxyethyloctylsulfoxide (n-Octyl-2-hydroxyethyl sulfoxide) P-1105
9
Non-ionic Detergents
Product Number
Product
CMC (mM)
Conditions
N (H2O)
References
P-1060
MEGA-8
73
25°C
F. Yu and R.E. McCarty, Arch. Biochem. Biophys. 238, 61 (1985)
P-1165
MEGA-9
25
25°C
V. de Pinto et al. , Eur. J. Biochem. 183, 179 (1989)
50
15°C
P-1000
MEGA-10
7
P-1175
MEGA-12
0.35
25°C
Y.-P. Zhu et al., J. Surf. Det. 2, 357 (1999)
P-1150
n-Nonyl β-Dglucopyranoside
6.5
low ionic strength 133
W.J. de Grip and P.H.M. Bovee-Geurts, Chem. Phys Lipids 23, 321 (1979)
3.5
1M NaCl
7.5
25°C
F. Yu and R.E. McCarty, Arch. Biochem. Biophys. 238, 61 (1985)
23.2
W.J. de Grip and P.H.M. Bovee-Geurts, Chem. Phys Lipids 23, 321 (1979)
13.5
low ionic strength 84 27-100 1M NaCl
25.0
25°C
K. Shinoda et al., Bull. Chem. Soc. Jpn. 34, 237 (1961)
P-1110
Octyl glucoside
M. Hanatani et al., J. Biochem. 95, 1349 (1984)
P-1170
n-Dodecyl-β-Dmaltoside
170
78-149 98
P-1095
n-Hexyltrioxyethylene
100
25°C
P-1085
n-Hexyltetraoxyethylene
90
20°C
P-1080
n-Hexylpentaoxyethylene
90
20°C
P-1050
Ethyleneglycolmonooctyl ether
4.9
25°C
P-1125
n-Octyltrioxyethylene
7.5
25°C
P-1120
n-Octyltetraoxyethylene
12.4
6°C
3.6
60°C
M. Corti et al., Phys. Rev. Lett. 48, 1617 (1982) R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986)
25°C
M. Zulauf and J.P. Rosenbusch, J. Phys. Chem. 87, 856 (1983)
P-1115
n-Octylpentaoxyethylene
4.3
P-1140
n-Octylpolyoxyethylene
6.6
P-1010
n-Decyltetraoxyethylene
0.98
10°C
0.68
25°C
P-1005
n-Decylpentaoxyethylene
1.18
10°C
0.81
25°C
P-1105
10
2-Hydroxyethyloctyl sulfoxide
29.9
W.J. de Grip, Methods Enzymol. 81, 256 (1982) J. Kern et al., Photosynth. Res. 84, 153 (2005) P. Becher, Micelle formation in aqueous and nonaqueous solutions. In: Nonionic Surfactants, M.J. Schick, ed., Marcel Dekker Inc., New York, p. 478 (1967)
82
R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986) 53 ± 10
P. Becher, Micelle formation in aqueous and nonaqueous solutions. In: Nonionic Surfactants, M.J. Schick, ed., Marcel Dekker Inc., New York, p. 478 (1967)
73
R.M. Garavito and J.P. Rosenbusch, Methods Enzymol. 125, 309 (1986)
PRODUCT BROCHURES AMYLOID PEPTIDES
ANTIMICROBIAL PEPTIDES
CALCITONIN GENE-RELATED PEPTIDES
CASPASE SUBSTRATES INHIBITORS
CYSTEINE DERIVATIVES
DAP AND DAB DERIVATIVES
DIABETES PEPTIDES
ENDOTHELINS
FRET SUBSTRATES
GHRELIN, LEPTIN AND OBESTATIN
1
1
LHRH AGONISTS AND ANTAGONISTS
MATRIX METALLOPROTEINASES
MELANOMA PEPTIDES
N-METHYLATED AMINO ACID DERIVATIVES
NEUROPEPTIDE Y
NON-IONIC DETERGENTS
ORTHOGONALITY OF PROTECTING GROUPS
PAR ACTIVATING PEPTIDES
PEPTIDE YY
PEPTIDES IN COSMETICS
SECRETASE SUBSTRATES INHIBITORS
VETERINARY PEPTIDES
VIP/PACAP
T
T S
PRION PEPTIDES
PSEUDOPROLINE DIPEPTIDES
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